Method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord

ABSTRACT

The present invention relates to a method of isolating a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, the method comprising cultivating umbilical cord tissue in a culture medium comprising DMEM (Dulbecco&#39;s modified eagle medium), F12 (Ham&#39;s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum). The invention also relates to a mesenchymal stem population isolated from the amniotic membrane of the umbilical cord, wherein at least about 90% or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105 and lack expression of the following markers: CD34, CD45 and HLA-DR. The invention also relates to a pharmaceutical composition of this mesenchymal stem population.

COPYRIGHT PROTECTION

A portion of the disclosure of this patent document contains materialwhich is subject to (copyright or mask work) protection. The (copyrightor mask work) owner has no objection to the facsimile reproduction byanyone of the patent document or the patent disclosure, as it appears inthe Patent and Trademark Office patent file or records, but otherwisereserves all (copyright or mask work) rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.Provisional Application No. 62/404,582, filed Oct. 5, 2016, the contentof which is hereby incorporated by reference it its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to a method of isolating mesenchymal stemcells (or such a stem cell population from the amniotic membrane ofumbilical cord, as well as a mesenchymal stem cell population isolatedfrom the amniotic membrane of the umbilical cord. The invention is alsodirected to a cell culture medium for isolating mesenchymal stem cellsfrom the amniotic membrane of the umbilical cord. The invention is alsodirected to a pharmaceutical composition and uses of the isolatedmesenchymal stem cell population. The invention is also directed tomethods of treating a disease or disorder comprising administering amesenchymal stem cell population or a pharmaceutical compositioncontaining such a mesenchymal stem cell population of the invention to asubject in need thereof.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells isolated from the amniotic membrane of theumbilical cord have been first reported in US patent application2006/0078993 (leading to granted U.S. Pat. Nos. 9,085,755 and 9,737,568)and the corresponding International patent application WO2006/019357.Since then, the umbilical cord tissue has gained attention as a sourceof multipotent cells; due to its widespread availability, the umbilicalcord and in particular stem cells isolated from the amniotic membrane ofthe umbilical cord (also referred to as “cord lining stem cells”) havebeen considered as an excellent alternative source of cells forregenerative medicine. See, Jeschke et al. Umbilical Cord LiningMembrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: theSimilarities and Differences; The Open Tissue Engineering andRegenerative Medicine Journal, 2011, 4, 21-27.

A subsequent study compared the phenotype, proliferation rate,migration, immunogenicity, and immunomodulatory capabilities of humanmesenchymal stem cells (MSCs) derived from the amniotic membrane of theumbilical cord (umbilical cord lining (CL-MSCs), umbilical cord blood(CB-MSCs), placenta (P-MSCs), and Wharton's jelly (WJ-MSCs) (Stubbendorfet al, Immunological Properties of Extraembryonic Human MesenchymalStromal Cells Derived from Gestational Tissue, STEM CELLS ANDDEVELOPMENT Volume 22, Number 19, 2013, 2619-2629. Stubbendorf et alconcluded that extraembryonic gestational tissue-derived MSC populationsshow a varied potential to evade immune responses as well as exertimmunomodulatory effects. The authors also found that CL-MSCs showed themost promising potential for a cell-based therapy, as the cells showedlow immunogenicity, but they also showed enhanced proliferative andmigratory potential so that future research should concentrate on thebest disease models in which CL-MSCs could be administered.

While mesenchymal stem cells of the amniotic membrane can easily beobtained using the protocol as described in US patent application2006/0078993 and International patent application WO2006/019357, itwould be of advantage for clinical trials with these cord lining MSC tohave at hand a method that allows to isolate a population of these cordlining MSC's that is highly homogenous and can thus be used for clinicaltrials.

Accordingly, it is an object of the invention to provide a method ofisolating a population of mesenchymal stem cells from the amnioticmembrane of umbilical cord that meets this need. It is thus also anobject of the invention to provide a highly homogenous population ofmesenchymal stem cells isolated from the amniotic membrane of theumbilical cord.

SUMMARY OF THE INVENTION

This object is accomplished by the methods, the mesenchymal stempopulation, the respective pharmaceutical composition and cell culturemedium having the features of the independent claims.

In a first aspect, the invention provides a method of isolating amesenchymal stem cell population from the amniotic membrane of theumbilical cord, the method comprising cultivating umbilical cord tissuein a culture medium comprising DMEM (Dulbecco's modified eagle medium),F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).

In a second aspect, the invention provides an isolated mesenchymal stempopulation of the amniotic membrane of the umbilical cord, wherein atleast about 90% or more cells of the stem cell population express eachof the following markers: CD73, CD90 and CD105. Preferably, the isolatedmesenchymal stem population lack expression of the following markers:CD34, CD45 and HLA-DR. In embodiments of this second aspect, at leastabout 91% or more, about 92% or more, about 92% or more, about 93% ormore, about 94% or more, about 95% or more, about 96% or more, about 97%or more, about 98% or more about 99% or more cells of the isolatedmesenchymal stem cell population express each of CD73, CD90 and CD105.In addition, in these embodiments of the second aspect, at least about91% or more, about 92% or more, about 92% or more, about 93% or more,about 94% or more, about 95% or more, about 96% or more, about 97% ormore, about 98% or more about 99% or more cells of the isolatedmesenchymal stem cell population preferably lack expression of themarkers CD34, CD45 and HLA-DR. The mesenchymal stem cell population maybe obtained by a method of isolating a mesenchymal stem cell populationof the first aspect.

In a third aspect, the invention provides a pharmaceutical compositioncontaining a mammalian cell of (the second aspect of) the invention.

In a fourth aspect, the invention provides a method of making a culturemedium for isolating the method comprising mixing to obtain a finalvolume of 500 ml culture medium:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) to obtain a final concentration of2.5% (v/v).

In a fifth aspect, the invention provides a cell culture mediumobtainable by the method of the fourth aspect.

In a sixth aspect, the invention provides a method of isolatingmesenchymal stem cells from the amniotic membrane of the umbilical cord,comprising cultivating amniotic membrane tissue in the culture mediumprepared by the method of the fourth aspect.

In a seventh aspect, the invention provides a cell culture mediumcomprising:

DMEM in the final concentration of about 55 to 65% (v/v),

F12 in a final concentration of about 5 to 15% (v/v),

M171 in a final concentration of about 15 to 30% (v/v) and

FBS in a final concentration of about 1 to 8% (v/v).

In an eight aspect, the invention provides the use of a cell culturemedium of the seventh aspect for the isolation of mesenchymal stem cellsfrom the amniotic membrane of umbilical cord.

In a ninth aspect, the invention provides the use of a cell culturemedium of the seventh aspect for the cultivation of mesenchymal stemcells from the amniotic membrane of umbilical cord.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the drawings, in which:

FIG. 1 shows the technical information sheet of Lonza for Dulbecco'smodified eagle medium, including the catalogue number of the DMEM usedfor the making of the illustrative example of a medium of the invention(PTT-6) in the Experimental Section;

FIG. 2 shows the technical information sheet of Lonza for Ham's F12medium;

FIG. 3 shows the technical information sheet of Lonza for DMEM:F12 (1:1)medium, including the catalogue number of the DMEM:F12 (1:1) medium usedfor the making of the illustrative example of a medium of the invention(PTT-6) in the Experimental Section;

FIG. 4 shows the technical information sheet of Life TechnologiesCorporation for M171 medium, including the catalogue number of the M171mediu used for the making of the illustrative example of a medium of theinvention (PTT-6) in the Experimental Section;

FIG. 5 shows the list of ingredients, including their commercialsupplier and the catalogue number that have been used in theExperimental Section for the making of the medium PTT-6.

FIGS. 6a-6c show the results of flow cytometry experiments in whichmesenchymal stem cells isolated from the umbilical cord have beenanalysed for the expression of the mesenchymal stem cell markers CD73,CD90 and CD105. For these experiments, mesenchymal stem cells wereisolated from umbilical cord tissue by cultivation of the umbilical cordtissue in three different cultivation media, followed by subculturing ofthe mesenchymal stem cells in the respective medium. The three followingculture media were used in these experiments: a) 90% (v/v/DMEMsupplemented with 10% FBS (v/v), b) the culture medium PTT-4 describedin US patent application 2006/0078993 and the correspondingInternational patent application WO2006/019357 that consist of 90% (v/v)CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of WO2006/019357 andc) the culture medium of the present invention PPT-6 the composition ofwhich is described herein. In this flow cytometry analysis, twodifferent samples of the cord lining mesenchymal stem cell (CLMC)population were analysed for each of the three used culture media. Theresults are shown in FIG. 6a to FIG. 6 c.

FIG. 6a shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in DMEM/10% FBS.

FIG. 6b shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-4.

FIG. 6c shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-6.

FIGS. 7a-7b show the results of flow cytometry experiments in whichmesenchymal stem cells isolated from the umbilical cord have beenanalysed for their expression of stem cells markers (CD73, CD90 andCD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen—antigen D Related)that are used for defining the suitability of multipotent humanmesenchymal stem cells for cellular therapy and compared to theexpression of these markers by bone marrow mesenchymal stem cells. Forthis experiment, the mesenchymal stem cells of the amniotic membrane ofthe umbilical cord were isolated from umbilical cord tissue bycultivation of the umbilical cord tissue in the culture medium of thepresent invention PPT-6 while the bone marrow mesenchymal stem cellswere isolated from human bone marrow using a standard protocol.

FIG. 7a shows the percentage of isolated mesenchymal cord lining stemcells that express the stem cell markers CD73, CD90 and CD105 and lackexpression of CD34, CD45 and HLA-DR after isolation from umbilical cordtissue and cultivation in PTT-6 medium.

FIG. 7b shows the percentage of isolated bone marrow mesenchymal stemcells that express CD73, CD90 and CD105 and lack expression of CD34,CD45 and HLA-DR.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, in a first aspect the invention is directed to amethod of isolating a mesenchymal stem cell population from the amnioticmembrane of the umbilical cord, the method comprising cultivatingumbilical cord tissue in a culture medium comprising DMEM (Dulbecco'smodified eagle medium), F12 (Ham's F12 Medium), M171 (Medium 171) andFBS (Fetal Bovine Serum). It has been surprisingly found in the presentapplication that using such a medium provides for the isolation of amesenchymal stem cell population from the amniotic membrane of theumbilical cord of which more than 90%, or even 99% or more of the cellsare positive for the three mesenchymal stem cell markers CD73, CD90 andwhile at the same these stem cells lack expression of CD34, CD45 andHLA-DR (see the Experimental Section), meaning 99% or even more cells ofthis population express the stem cell markers CD73, CD90 and CD105 whilenot expressing the markers CD34, CD45 and HLA-DR. Such an extremelyhomogenous and well defined cell population is the ideal candidate forclinical trials and cell based therapies since, they for example, fullymeet the criteria generally accepted for human mesenchymal stem cells tobe used for cellular therapy as defined, for example, by Dominici et al,“Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement”,Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al, “Production ofmesenchymal stromal/stem cells according to good manufacturingpractices: a, review”, Stem Cell Research & Therapy 2013, 4:66), Vonk etal., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta MedicaLituanica. 2012. Vol. 19. No. 2. P. 75-79. Also, using a bioreactor suchas a Quantum Cell Expansion System, it is possible to obtain highnumbers of mesenchymal stem cells such as 300 to 700 million mesenchymalstem cells per run (see also the Experimental Section). Thus, thepresent invention allows to provide the amounts of stem cells that areneeded for therapeutic applications such as their use in wound healingin a cost efficient manner. In addition, all components used for makingthe culture medium of the present invention are commercially availablein GMP quality. Accordingly, the present invention opens the route tothe GMP production of this highly homogenous mesenchymal stem cellpopulation from the amniotic membrane of the umbilical cord.

In this context, it is noted that the culture medium of the presentinvention allows the isolation of a mesenchymal stem cell population(also referred hereas as “mesenchymal stem cells”) from the amnioticmembrane under conditions that allow cell proliferation of themesenchymal stem/progenitor cells without differentiation of themesenchymal stem/progenitor cells. Thus, after isolation of themesenchymal stem cells from the amniotic membrane as described hereinthe isolated mesenchymal stem/progenitor cell population has thecapacity to differentiate into multiple cell types as described in USpatent application 2006/0078993, U.S. Pat. No. 9,085,755, Internationalpatent application WO2006/019357, U.S. Pat. No. 8,287,854 orWO2007/046775, for instance. As described in US patent application2006/0078993, for example, the mesenchymal stem cells of the amnioticmembrane of the umbilical cord have a spindle shape, express thefollowing genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), andsecrete Activin A and Follistatin. The mesenchymal stem cells isolatedin the present invention can, for example, be differentiated into anytype of mesenchymal cell such as, but not limited to, adipocyte, skinfibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts,cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes,mucin producing cells, cells derived from endocrine glands such asinsulin producing cells (for example, β-islet cells) or neurectodermalcells. The stem cells isolated in the present invention can bedifferentiated in vitro in order to subsequently use the differentiatedcell for medical purposes. An illustrative example of such an approachis the differentiation of the mesenchymal stem cells into insulinproducing β-islet cells which can then be administered, for example byimplantation, to a patient that suffers from an insulin deficiency suchas diabetes mellitus (cf. also WO2007/046775 in this respect).Alternatively, the mesenchymal stem cells of the invention can be usedin their undifferentiated state for cell based therapy, for example, forwound healing purposes such as treatment of burns or chronic diabeticwounds. In these therapeutic applications the mesenchymal stem cells ofthe invention can either serve to promote wound healing by interactingwith the surrounding diseased tissue or can also differentiate into arespective skin cell (cf., again WO2007/046775, for example).

In this context, it is noted that the mesenchymal stem cell populationdescribed herein can be isolated and cultivated (i.e. are derived) fromany umbilical cord tissue as long as the umbilical cord tissue containsthe amniotic membrane (which is also referred to as “cord lining”).Accordingly, the mesenchymal stem cell population can be isolated from(pieces of) the entire umbilical cord as described in the Experimentalsection of the present application. This umbical cord tissue may thuscontain, in addition to the amniotic membrane, any othertissue/component of the umbilical cord. As shown, for example, in FIG.16 of US patent application 2006/0078993 or International patentapplication WO2006/019357, the amniotic membrane of the umbilical cordis the outmost part of the umbilical cord, covering the cord. Inaddition, the umbilical cord contains one vein (which carriesoxygenated, nutrient-rich blood to the fetus) and two arteries (whichcarry deoxygenated, nutrient-depleted blood away from the fetus). Forprotection and mechanical support these three blood vessels are embeddedin the Wharton's jelly, a gelatinous substance made largely frommucopolysaccharides. Accordingly, the umbilical cord tissue used in thepresent invention can also comprise this one vein, the two arteries andthe Wharton's jelly. The use of such an entire (intact) section of theumbilical cord has the advantage that the amniotic membrane does notneed to be separated from the other components of the umbilical cord.This reduces the isolation steps and thus makes the method of thepresent invention, simpler, faster, less error prone and moreeconomical—which are all important aspects for the GMP production thatis necessary for therapeutic application of the mesenchymal stem cells.The isolation of the mesenchymal stem cells can thus start by tissueexplant, which may be followed by subsequent subculturing (cultivation)of the isolated mesenchymal stem cells if greater amounts of themesenchymal stem cells are desired, for example, for use in clinicaltrials. Alternatively, it is also possible to first separate theamniotic membrane from the other components of the umbilical cord andisolate the mesenchymal cord lining stem cells from the amnioticmembrane by cultivation of the amniotic membrane in a culture medium ofthe present invention. This cultivation can also be carried out bytissue explant, optionally followed by subculturing of the isolatedmesenchymal stem cells. In this context, the term “tissue explant” or“tissue explant method” is used in its regular meaning in the art torefer a method in which a tissue, once being harvested, or a piece ofthe tissue is being placed in a cell culture dish containing culture(growth) medium and by which over time, the stem cells migrate out ofthe tissue onto the surface of the dish. These primary stem cells canthen be further expanded and transferred into fresh dishes throughmicropropagation (subculturing) as also described here. In this context,it is noted that in terms of production of the cells for therapeuticpurposes, in the first step of isolating the amniotic membranemesenchymal stem cells from the umbilical cord, a master cell bank ofthe isolated mesenchymal stem cells is obtained, while the subsequentsubculturing a working cell bank can be obtained. If a mesenchymal stemcell population of the invention (in particular a population of themesenchymal stem cells of which at least about 98% or 99% or expresseach of the markers CD73, CD90 and CD105 and lack expression of each ofthe markers: CD34, CD45 and HLA-DR) is used for clinical trials or as anapproved therapeutic, a cell population of the working cell bank will betypically used for this purpose. Both the stem cell population of theisolation step (which may make up the master cell bank) and the stemcell population of the subculturing step (which may make up the workingcell bank) can, for example, be stored in cryo-preserved form.

As mentioned above, the present method of isolating mesenchymal stemcells from the amniotic membrane of umbilical cord has the advantagethat all components used in the culture medium of the invention areavailable in GMP quality and thus provide the possibility to isolate themesenchymal stem cells under GMP conditions for subsequent therapeuticadministration.

By “DMEM” is meant Dulbecco's modified eagle medium which was developedin 1969 and is a modification of basal medium eagle (BME) (cf. FIG. 1showing the data sheet of DMEM available from Lonza). The original DMEMformula contains 1000 mg/L of glucose and was first reported forculturing embryonic mouse cells. DMEM has since then become a standardmedium for cell culture that is commercially available from varioussources such as ThermoFisher Scientific (catalogue number 11965-084),Sigma Aldrich (catalogue number D5546) or Lonza, to new only a fewsuppliers. Thus, any commercially available DMEM can be used in thepresent invention. In preferred embodiments, the DMEM used herein is theDMEM medium available from Lonza under catalog number 12-604F. Thismedium is DMEM supplemented with 4.5 g/L glucose and L-glutamine). Inanother preferred embodiment the DMEM used herein is the DMEM medium ofSigma Aldrich catalogue number D5546 that contains 1000 mg/L glucose,and sodium bicarbonate but is without L-glutamine.

By “F12” medium is meant Ham's F12 medium. This medium is also astandard cell culture medium and is a nutrient mixture initiallydesigned to cultivate a wide variety of mammalian and hybridoma cellswhen used with serum in combination with hormones and transferrin (cf.FIG. 2, showing the data sheet of Ham's F12 medium from Lonza). Anycommercially available Ham's F12 medium (for example, from ThermoFisherScientific (catalogue number 11765-054), Sigma Aldrich (catalogue numberN4888) or Lonza, to new only a few suppliers) can be used in the presentinvention. In preferred embodiments, Ham's F12 medium from Lonza isused.

By “DMEM/F12” or “DMEM:F12” is meant a 1:1 mixture of DMEM with Ham'sF12 culture medium (cf. FIG. 3 showing the data sheet for DMEM:F12 (1:1)medium from Lonza). Also DMEM/F12 (1:1) medium is a widely used basalmedium for supporting the growth of many different mammalian cells andis commercially available from various supplier such as ThermoFisherScientific (catalogue number 11330057), Sigma Aldrich (catalogue numberD6421) or Lonza. Any commercially available DMEM:F12 medium can be usedin the present invention. In preferred embodiments, the DMEM:F12 mediumused herein is the DMEM/F12 (1:1) medium available from Lonza undercatalog number 12-719F (which is DMEM:F12 with L-glutamine, 15 mM HEPES,and 3.151 g/L glucose).

By “M171” is meant culture medium 171, which has been developed as basalmedium for the culture of for the growth of normal human mammaryepithelial cells (cf. FIG. 4 showing the data sheet for M171 medium fromLife Technologies Corporation). Also this basal medium is widely usedand is commercially available from supplier such as ThermoFisherScientific or Life Technologies Corporation (catalogue number M171500),for example. Any commercially available M171 medium can be used in thepresent invention. In preferred embodiments, the M171 medium used hereinis the M171 medium available from Life Technologies Corporation undercatalogue number M171500.

By “FBS” is meant fetal bovine serum (that is also referred to as “fetalcalf serum”), i.e. the blood fraction that remains after the naturalcoagulation of blood, followed by centrifugation to remove any remainingred blood cells. Fetal bovine serum is the most widely usedserum-supplement for in vitro cell culture of eukaryotic cells becauseit has a very low level of antibodies and contains more growth factors,allowing for versatility in many different cell culture applications.The FBS is preferably obtained from a member of the International SerumIndustry Association (ISIA) whose primary focus is the safety and safeuse of serum and animal derived products through proper origintraceability, truth in labeling, and appropriate standardization andoversight. Suppliers of FBS that are ISIA members include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GEHealthcare, Gibco by Thermo Fisher Scientific and Life ScienceProduction, to mention only a few. In currently preferred embodiments,the FBS is obtained from GE Healthcare under catalogue number A15-151.

Turning now to the culture medium of the present invention, the culturemedium may comprise for the isolation or cultivation of the mesenchymalcord lining stem cells DMEM in a final concentration of about 55 to 65%(v/v), F12 in a final concentration of about 5 to 15% (v/v), M171 in afinal concentration of about 15 to 30% (v/v) and FBS in a finalconcentration of about 1 to 8% (v/v). The value of “% (v/v)” as usedherein refers to the volume of the individual component relative to thefinal volume of the culture medium. This means, if DMEM is, for example,present in the culture medium a final concentration of about 55 to 65%(v/v), 1 liter of culture medium contains about 550 to 650 ml DMEM.

In other embodiments, the culture medium may comprise DMEM in a finalconcentration of about 57.5 to 62.5% (v/v), F12 in a final concentrationof about 7.5 to 12.5% (v/v), M171 in a final concentration of about 17.5to 25.0% (v/v) and FBS in a final concentration of about 1.75 to 3.5%(v/v). In further embodiments, the culture medium may comprise DMEM in afinal concentration of about 61.8% (v/v), F12 in a final concentrationof about 11.8% (v/v), M171 in a final concentration of about 23.6% (v/v)and FBS in a final concentration of about 2.5% (v/v).

In addition to the above-mentioned components, the culture medium maycomprise supplements that are advantages for cultivation of themesenchymal cord lining stem cells. The culture medium of the presentinvention may, for example, comprises Epidermal Growth Factor (EGF). Ifpresent, EGF may be present in the culture medium in a finalconcentration of about 1 ng/ml to about 20 ng/ml. In some of theseembodiments, the culture medium may comprise EGF in a finalconcentration of about 10 ng/ml.

The culture medium of the present invention may also comprises insulin.If present, insulin may be present in a final concentration of about 1μg/ml to 10 μg/ml. In some of these embodiments, the culture medium maycomprise Insulin in a final concentration of about 5 μg/ml.

The culture medium may further comprises at least one of the followingsupplements: adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyroninesodium salt (T3). In such embodiments, the culture medium may compriseall three of adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyroninesodium salt (T3). In these embodiments, the culture medium may comprisesmay comprise adenine in a final concentration of about 0.05 to about 0.1μg/ml adenine, hydrocortisone in a final concentration of about 1 toabout 10 μg/ml hydrocortisone and/or 3,3′,5-Triiodo-L-thyronine sodiumsalt (T3) in a final concentration of about 0.5 to about 5 ng/ml.

In the method of the invention, the umbilical cord tissue may becultured till a suitable number of (primary) mesenchymal cord liningstem cells have outgrown from the tissue. In typical embodiments, theumbilical cord tissue is cultivated until cell outgrowth of themesenchymal stem cells of the amniotic membrane reaches about 70 toabout 80% confluency. It is noted here that the term “confluency” or“confluence” is used in its regular meaning in the art of cell cultureand is meant as an estimate/indicator of the number of adherent cells ina culture dish or a flask, referring to the proportion of the surfacewhich is covered by cells. For example, 50 percent confluence meansroughly half of the surface is covered and there is still room for cellsto grow. 100 percent confluence means the surface is completely coveredby the cells, and no more room is left for the cells to grow as amonolayer.

Once a suitable number of primary cells (mesenchymal cord lining stemcells) have been obtained from the cord lining tissue by tissue explant,the mesenchymal stem cells are removed from the cultivation containerused for the cultivation. By so doing, a master cell bank containing the(primary) isolated mesenchymal stem cells of the amniotic membrane canbe obtained. Typically, since mesenchymal stem cells are adherent cells,removing is carried out using standard enzymatic treatment. For example,the enzymatic treatment may comprise trypsination as described inInternational US patent application 2006/0078993, International patentapplication WO2006/019357 or International patent applicationWO2007/046775, meaning outgrowing cells can be harvested bytrypsinization (0.125% trypsin/0.05% EDTA) for further expansion. If theharvested mesenchymal stem cells are, for example, used for generating amaster cell bank, the cells can also be cryo-preserved and stored forfurther use as explained herein below.

Once being harvested, the mesenchymal stem cells can be transferred to acultivation container for subculturing. The subculturing can also bestarted from frozen primary cells, i.e. from the master cell bank. Forsubculturing any suitable amount of cells can be seeded in a cultivationcontainer such as cell culture plate. The mesenchymal cells can, forthis purpose, be suspended in a suitable medium (most conveniently, theculture medium of the present invention) for subculturing at aconcentration of, for example, about 0.5×10⁶ cells/ml to about 5.0×10⁶cells/ml. In one embodiment the cells are suspended for subcultivationat a concentration of about 1.0×10⁶ cells/ml. The subculturing can becarried by cultivation either in simple culture flasks but also, forexample, in a multilayer system such as CellStacks (Corning, Corning,N.Y., USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc.,Waltham, Mass., USA) that can be stacked in incubators. Alternatively,the subculturing can also be carried out in a closed self-containedsystem such as a bioreactor. Different designs of bioreactors are knownto the person skilled in the art, for example, parallel-plate,hollow-fiber, or micro-fluidic bioreactors. See, for example, Sensebe etal. “Production of mesenchymal stromal/stem cells according to goodmanufacturing practices: a review”, supra. An illustrative example of acommercially hollow-fiber bioreactor is the Quantum® Cell ExpansionSystem (Terumo BCT, Inc). that has, for example, been used for theexpansion of bone marrow mesenchymal stem cells for clinical trials(cf., Hanley et al, Efficient Manufacturing of Therapeutic MesenchymalStromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014August; 16(8): 1048-1058). Another example of a commercially availablebioreactors that can be used for the subculturing of the mesenchymalstem cell population of the present invention is the Xuri Cell ExpansionSystem available from GE Healthcare. The cultivation of the mesenchymalstem cell population in an automated system such as the Quantum® CellExpansion System is of particular benefit if a working cell bank fortherapeutic application is to be produced under GMP conditions and ahigh number of cells is wanted.

The subculturing of the mesenchymal cord ling stem cells of theinvention takes place in in a culture medium of the present invention.Accordingly, the culture medium of the present invention can be usedboth for the isolation of the mesenchymal stem cells from the amnioticmembrane and the subsequent cultivation of the isolated primary cells bysubcultivation. Also for the subcultivation, the mesenchymal stem cellscan be cultured till a suitable amount of cells have grown. Inillustrative embodiments the mesenchymal stem cells are subcultured tillthe mesenchymal stem cells reach about 70 to about 80% confluency.

The isolation/cultivation of the population of mesenchymal cord liningstem cells can be carried out under standard condition for thecultivation of mammalian cells. Typically, the method of the inventionof isolating the population of the mesenchymal cord lining stem cells istypically carried out at conditions (temperature, atmosphere) that arenormally used for cultivation of cells of the species of which the cellsare derived. For example, human umbilical cord tissue and themesenchymal cord lining stem cells, respectively, are usually cultivatedat 37° C. in air atmosphere with 5% CO₂. In this context, it is notedthat the in present invention the mesenchymal cells may be derived ofany mammalian species, such as mouse, rat, guinea pig, rabbit, goat,horse, dog, cat, sheep, monkey or human, with mesenchymal stem cells ofhuman origin being preferred in one embodiment.

Once a desired/suitable number of mesenchymal cord lining stem cellshave been obtained from the subculture, the mesenchymal stem cells areharvested by removing them from the cultivation container used for thesubcultivation. The harvesting of the mesenchymal stem cells istypically again carried out by enzymatic treatment, including comprisestrypsination of the cells. The isolated mesenchymal stem cells aresubsequently collected and are either be directedly used or preservedfor further use. Typically, preserving is carried out bycryo-preservation. The term “cryo-preservation” is used herein in itsregular meaning to describe a process where the mesenchymal stem cellsare preserved by cooling to low sub-zero temperatures, such as(typically) −80° C. or −196° C. (the boiling point of liquid nitrogen).Cryo-preservation can be carried out as known to the person skilled inthe art and can include the use of cryo-protectors such asdimethylsulfoxide (DMSO) or glycerol, which slow down the formation ofice-crystals in the cells of the umbilical cord.

The isolated population of the mesenchymal cord lining stem cells thatis obtained by the isolation method of the present invention is highlydefined and homogenous. In typical embodiments of the method at leastabout 90% or more, about 91% or more, about 92% or more, about 92% ormore, about 93% or more, about 94% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more about 99% or more of theisolated mesenchymal stem cells express the following markers: CD73,CD90 and CD105. In addition, in these embodiments at least about 90% ormore, about 91% or more, about 92% or more, about 92% or more, about 93%or more, about 94% or more, about 95% or more, about 96% or more, about97% or more, about 98% or more about 99% or more of the isolatedmesenchymal stem cells may lack expression of the lack expression of thefollowing markers: CD34, CD45 and HLA-DR. In particular embodiments,about 97% or more, about 98% or more, or about 99% or more of theisolated mesenchymal stem cell population express CD73, CD90 and CD105while lacking expression of CD34, CD45 and HLA-DR.

Thus, in line with the above disclosure the present invention is alsodirected to a mesenchymal stem population isolated from the amnioticmembrane of the umbilical cord, wherein at least about 90% or more cellsof the stem cell population express each of the following markers: CD73,CD90 and CD105. In preferred embodiments at least about 91% or more,about 92% or more, about 92% or more, about 93% or more, about 94% ormore, about 95% or more, about 96% or more, about 97% or more, about 98%or more about 99% or more cells of the isolated mesenchymal stem cellpopulation are CD73+, CD90+ and CD105+, meaning that this percentage ofthe isolate cell population express each of CD73, CD90 and CD105 (cf.the Experimental Section of the present application). In addition, atleast about 90% or more, about 91% or more, about 92% or more, about 92%or more, about 93% or more, about 94% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more about 99% or more ofthe isolated mesenchymal stem cells may lack expression of the lackexpression of the following markers. In particular embodiments about 97%or more, about 98% or more, or about 99% or more of the isolatedmesenchymal stem cell population express CD73, CD90 and CD105 whilelacking expressing of CD34, CD45 and HLA-DR. Such a highly homogenouspopulation of mesenchymal stem cells derived from the amniotic membraneof the umbilical cord has been reported here for the first time andmeets the criteria for mesenchymal stem cells to be used for cellulartherapy (also cf. the Experimental Section and, for example, Sensebe etal. “Production of mesenchymal stromal/stem cells according to goodmanufacturing practices: a review”, supra). It is noted in this contextthat this mesenchymal stem cell population can be obtained by either theisolating method of the present invention but also by a different methodsuch as cell sorting, if wanted.

In line with the above, the present invention is also directed to apharmaceutical composition comprising a mesenchymal stem populationisolated from the amniotic membrane of the umbilical cord, wherein atleast about 90% or more cells of the stem cell population express eachof the following markers: CD73, CD90 and CD105 and optionally, lackexpression of CD34, CD45 and HLA-DR. The pharmaceutical composition maycomprise any pharmaceutically acceptable excipient and may be formulatedfor any desired pharmaceutical way of administration. The pharmaceuticalcomposition may, for example, be adapted for systemic or topicalapplication.

In a further aspect the invention is directed to a method of making aculture medium for isolating the method comprising, mixing to obtain afinal volume of 500 ml culture medium:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of2.5% (v/v).

As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham'sF12 medium. Thus, 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 mlF12. Accordingly, when using this method of making a culture medium, thefinal concentrations (v/v) mit 500 ml total volume are as follows:

DMEM: 250 ml+59 ml=309 ml, corresponds to 309/500=61.8% (v/v)

M171: 118 ml, corresponds to 118/500=23.6% (v/v)

F12: 59 ml, corresponds to 59/500=11.8% (v/v).

Embodiments of this method of making a culture medium further compriseadding

v. 1 ml EGF stock solution (5 μg/ml) to achieve a final EGFconcentration of 10 ng/ml, and

vi. Insulin 0.175 ml stock solution (14.28 mg/ml) to achieve a finalinsulin concentration of 5 μg/ml.

It is noted here that in these embodiments, the above-mentioned volumesof these components i. to vi when result in a final volume of 499.675 mlculture medium. If no further components are added to the culturemedium, the remaining 0.325 ml (to add up to a volume of 500 ml) can,for example, be any of components i. to iv, that means either DMEM,M171, DMEM/F12 or FBS. Alternatively, the concentration of the stocksolution of EGF or Insulin can of course be adjusted such that the totalvolume of the culture medium is 500 ml. In addition, it is also notedthat components i. to iv. do not necessarily have to be added in theorder in which they are listed but it is of course also possible to useany order to mix these components to arrive at the culture medium of thepresent invention. This means, that for example, M171 and DMEM/F12 canbe mixed together and then combined with DMEM and FBS to reach finalconcentrations as described here, i.e. a final concentration of DMEM ofabout 55 to 65% (v/v), a final concentration of F12 of about 5 to 15%(v/v), a final concentration of M171 of about 15 to 30% (v/v) and afinal concentration of FBS of about 1 to 8% (v/v).

In other embodiments, the method further comprises adding to DMEM avolume of 0.325 ml of one or more of the following supplements: adenine,hydrocortisone, 3,3′,5-Triiodo-L-thyronine sodium salt (T3), therebyreaching a total volume of 500 ml culture medium. In this embodiments,the final concentration of these supplements in DMEM may be as follows:

about 0.05 to 0.1 μg/ml adenine, for example about 0.025 μg/ml adenine,

about 1 to 10 μg/ml hydrocortisone,

about 0.5 to 5 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3), forexample 1.36 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3).

In line with the above disclosure, the invention is also directed to acell culture medium that is obtainable or that is obtained by the methodof making the medium as described here.

In addition, the invention also concerns a method of isolatingmesenchymal stem cells from the amniotic membrane of the umbilical cord,wherein this method comprises cultivating amniotic membrane tissue inthe culture medium prepared by the method as described here.

Thus, the present invention is also directed to a cell culture mediumcomprising:

DMEM in the final concentration of about 55 to 65% (v/v),

F12 in a final concentration of about 5 to 15% (v/v),

M171 in a final concentration of about 15 to 30% (v/v) and

FBS in a final concentration of about 1 to 8% (v/v).

In certain embodiments of the culture medium described here, the mediumcomprises DMEM in the final concentration of about 57.5 to 62.5% (v/v),F12 in a final concentration of about 7.5 to 12.5% (v/v), M171 in afinal concentration of about 17.5 to 25.0% (v/v) and FBS in a finalconcentration of about 1.75 to 3.5% (v/v). In other embodiments theculture medium may comprise DMEM in a final concentration of about 61.8%(v/v), F12 in a final concentration of about 11.8% (v/v), M171 in afinal concentration of about 23.6% (v/v) and FBS in a finalconcentration of about 2.5% (v/v).

In addition, the culture medium may further comprise Epidermal GrowthFactor (EGF) in a final concentration of about 1 ng/ml to about 20ng/ml. In certain embodiments, the culture medium comprise EGF in afinal concentration of about 10 ng/ml. The culture medium describedherein may further comprise Insulin in a final concentration of about 1μg/ml to 10 μg/ml. In such embodiments the culture medium may compriseInsulin in a final concentration of about 5 μg/ml.

The cell culture medium of the invention may further comprise at leastone of the following supplements: adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3). In certain embodiments theculture medium comprises all three of adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3). If present, the culturemedium may comprise adenine in a final concentration of about 0.01 toabout 0.1 μg/ml adenine or of about 0.05 to about 0.1 mg/ml adenine,hydrocortisone in a final concentration of about 0.1 to about 10 mg/mlhydrocortisone or of about 1 to about 10 mg/ml hydrocortisone and/or3,3′,5-Triiodo-L-thyronine sodium salt (T3) in a final concentration ofabout 0.5 to about 5 ng/ml.

In embodiments of the cell culture medium, 500 ml of the cell culturemedium of the present invention comprise:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) (final concentration of 2.5%)

In further embodiments, the cell culture medium may further comprise

v. EGF in a final concentration of 10 ng/ml, and

vi. Insulin in a final concentration of 5 μg/ml.

Both, insulin and and EGF can be added to to the culture medium using astock solution of choice, such that the total volume of the culturemedium does not exceed 500 ml.

In a particular example, the components i. to vi. of the culture mediumof the present invention are the components indicated in FIG. 5, meaningthey are obtained from the respective manufacturers using the cataloguenumber indicated in FIG. 5. The medium that is obtained from mixing thecomponents i. to vi. as indicated in FIG. 5 is also referred herein as“PTT-6”. It is again noted in this context that the constituents i. tovi. as well as any other ingredient such as an antibiotic of any othercommercial supplier can be used in making the medium of the presentinvention.

In addition, the cell culture medium of the invention may compriseadenine in a final concentration of about 0.01 to about 0.1 μg/mladenine or of about 0.05 to about 0.1 μg/ml adenine, hydrocortisone in afinal concentration of about 0.1 to 10 μg/ml, of about 0.5 to about 10μg/ml, or of about 1 to about 10 μg/ml hydrocortisone and/or3,3′,5-Triiodo-L-thyronine sodium salt (T3) in a final concentration ofabout 0.1 to about 5 ng/ml or of about 0.5 to about 5 ng/ml.

Finally, the invention also provides a method of treating a patienthaving a disease, the method comprising administering to the patient amesenchymal cord lining stem cell or a pharmaceutical compositioncontaining a stem cell as disclosed herein. The disease can be anydisease than described above. For treating the subject, the mesenchymalstem cell population of the invention may be administered in anysuitable way, for example, including but not limited to, topicaladministration, by implantation or by injection. The stem cellpopulation may, for example, be placed directly onto a wound such as aburn or a diabetic wound (see International patent applicationWO2007/046775). Alternatively, the stem cell population may also beimplanted subcutaneously, for example, directly under the skin, in bodyfat or the peritoneum.

The invention will be further illustrated by the following non-limitingExperimental Examples.

EXPERIMENTAL EXAMPLES

1. Cryopreservation of Umbilical Cord Tissue Prior to Isolation ofMesenchymal Stem Cells

Umbilical cord tissue (the umbilical cords were donated with informedconsent of the mother) was processed for the subsequent isolation of themesenchymal stel cells from the amniotic membrane of the umbilical cordas follows.

1.1 Washing of Umbilical Cord Tissue Sample:

a. Remove scalpels from the protective cover.

b. Hold the umbilical cord securely using the forceps and cut the cordinto a 10 cm length piece using a scalpel. Place the unusable cord backin the original tissue cup.

c. Transfer the 10 cm long umbilical cord piece into a new 150 mmculture dish. The 150 mm culture dish may be used in place of the cups.

d. Use the cover of the 150 mm culture dish as a resting place forforceps and scalpel.

e. Remove 25 ml Plasmalyte A (Baxter, Catalog #2B2543Q) with a 30 mlsyringe. Hold the syringe at a 45° angle using one hand and dispense thePlasmalyte A directly onto the umbilical cord tissue.

f. Holding the culture dish at a slight angle remove the Plasmalyte Awith a 30 ml syringe and blunt needle.

g. Collect used Plasmalyte A in a 300 ml transfer bag that serves as atrash container and dispose it in the biohazard bin.

h. Repeat wash procedure, if necessary using a new culture dish for eachwash. Make sure all blood clots on the surface have been removed. MorePlasmalyte A can be used if needed to clean the tissue.

i. Place the tissue into a new labeled tissue culture dish to continuecutting the tissue. Place 20 ml of Plasmalyte A into the dish so thetissue does not dry out while cutting it.

j. Cut the cords into equal approximately 1-cm sections resulting in 10sections in total.

k. Further cut each 1 cm section into smaller pieces with approximately0.3 cm×0.3 cm to 0.5 cm×0.5 cm per section.

l. Remove any Plasmalyte A that is in the dish.

m. Pull 25 ml Plasmalyte A with a 30 ml syringe from the originalPlasmalyte A bag and dispense directly on the umbilical cord tissuepieces.

n. Hold culture dish in an angle to collect all Plasmalyte A used forwashing the tissue on one side and remove it with a syringe and bluntneedle.

o. Repeat wash one more time. There should not be any clots left.

NOTE: If the cord is not frozen right away, the umbilical cord tissue iskept in Plasmalyte A until ready to freeze.

1.2 Cryopreservation of Umbilical Cord Tissue:

a. Prepare cryopreservation solution:

i. Prepare 50 ml freezing solution consisting of 60% Plasmalyte A, 30%of 5% Human Serum Albumin, and 10% dimethyl sulfoxide (DMSO).

ii. Label a 150 ml transfer bag with “Tissue freeze solution” and attacha plasma transfer set to the port using aseptic technique.

iii. Remove 30 ml Plasmalyte A with a 30 ml Syringe from the originalPlasmalyte A bag and transfer it in the transfer bag labeled “tissuefreeze solution” with the time and date solution is made.

iv. Remove 15 ml of 5% Human Serum Albumin with a 20 ml syringe andtransfer it into the labeled transfer bag.

v. Add 5 ml DMSO to the transfer bag.

vi. Mix well and record mixing of freeze solution

b. Remove the Plasmalyte A from the tissue before adding the freezesolution.

c. Using a 60 ml syringe, pull all 50 mls of the freeze solution intothe syringe add approximately 30 ml freeze solution to the 150 mm cellculture dish containing the umbilical cord tissue. Place a blunt needleon the syringe to keep it sterile.

d. Swirl the culture dish containing the tissue and freezing solutionevery minute for 10 minutes.

e. Using forceps, select 8 randomly chosen sections and place them ineach of the four 4 ml cryovials. Select 4 randomly chosen sections andplace them into one 1.8 ml cryovial. These sections should be free ofblood clots.

f. Fill each cryovial containing the umbilical cord tissue with theremaining freezing solution to the 3.6 ml filling line for the 4 mltubes and the 1.8 ml line for the 1.8 ml Nunc vial.

g. Label one Bactec Lytic/10-Anaerobic/F and one Bactec Plus Aerobic/Fbottle with tissue ID.

h. Remove 20 ml freeze solution from the culture dish with a syringe anda blunt needle, after wiping the Bactec vials with an alcohol swab,switch the blunt needle for an 18g needle and inoculate the aerobic andthe anaerobic Bactec bottles with 10 ml each.i. Start controlled rate freezer.j. After controlled rate freeze is completed place the units in acontinuous temperature monitored liquid nitrogen freezer until furtheruse.

2. Isolation of Mesenchymal Cord Lining Stem Cells from Umbilical CordTissue

2.1 Preparing Media for Processing MSCs from Umbilical Cord Tissue:

a. To make 500 ml PTT6 (culture/growth media) add the following in theorder listed:

i. DMEM, 250 ml

ii. M171 118 ml

iii. DMEM F12 118 ml

iv. FBS 12.5 ml (final concentration of 2.5%)

v. EGF 1 ml (final concentration of 10 ng/ml)

vi. Insulin 0.175 ml (final concentration of 5 μg/ml)

The above-mentioned volumes of components i. to vi when result in afinal volume of 499.675 ml culture medium. If no further components areadded to the culture medium, the remaining 0.325 ml (to add up to avolume of 500 ml) can, for example, be any of components i. to iv, thatmeans either DMEM, M171, DMEM/F12 or FBS. Alternatively, theconcentration of the stock solution of EGF or Insulin can of course beadjusted such that the total volume of the culture medium is 500 ml.Alternatively, a stock solution of an antibiotic such asPenicillin-Streptomycin-Amphotericin can be added to result in a finalvolume of 500 ml. It is also possible to add to the culture medium avolume of 0.325 ml of one or more of the following supplements: adenine,hydrocortisone, 3,3′,5-Triiodo-L-thyronine sodium salt (T3), therebyreaching a total volume of 500 ml culture medium.

vii. Label the bottle “PTT6” with date media was prepared, initial ofthe operator, and the phrase “expires on” followed by the expirationdate. Expiration date is the earliest expiration date of any of thecomponent or 1 month from the preparation date, whichever comes first.

b. To make the rinse media (Hank's Buffered Salt Solution (HBSS) withoutCalcium or Magnesium and with 5% FBS), add 2.5 ml FBS to 47.5 ml of HBSSin a 50 ml centrifuge tube. Label the tube “Rinse Media” with operatorinitials and date the media is made.

c. All media will be tested for sterility using BactecLytic/10-Anaerobic/F (Becton Dickinson & Company) and BactecPluc+Aerobic/F (Becton Dickinson & Company). Inject 20 ml of preparedmedia into each bottle.

2.2 Thawing of Umbilical Cord Tissue for MSC Harvesting:

a. Initiate the thaw once an operator is prepared to process the samplein the clean room. Do not thaw more than 1 vial at a time unless thevials originate from the same donor.

b. Wipe the water bath with disinfectant followed by 70% isopropanol andfill it with 1 L sterile water. Heat the water bath up to 36-38° C.

c. Prepare 10 mL of rinse medium consisting of 70% to 90% PlasmaLyte Ain the clean room under a biosafety cabinet. Sterile filter the solutionwith a 0.2-μm syringe filter attached to a 10 ml syringe and keep thesolution refrigerated until use.

d. Place a processing label on a 50 ml conical tube.

e. Confirm water bath temperature is at 36-38° C.

f. Take vial(s) of tissue from the liquid nitrogen storage and thawrapidly in the 37° C. water bath filled with 1 L of sterile water. Thevial holder for the Mr. Frosty Nalgene Cryo 1° C. freezing containerfloats with vials in place and can be used as a floating rack whenthawing samples.g. Remove the vial from the water bath and spray them with 70%Isopropanol solution. A good time to pull the vial from the water bathis when small ice can be seen floating in the vial—suggest internaltemperature of the vial is less than 37° C.h. Place vial into pass-through and alert the clean room processingtechnician.

2.3 Preparing for Tissue Processing:

a. Umbilical cord tissue processing should be performed in anenvironmentally monitored (EM) clean room: At the end of each shift,full room and hood cleaning are performed

b. Prepare/clean the biosafety cabinet.

c. Perform viable particle counting while working in the biosafetycabinet.

d. Assemble all necessary supplies in the biosafety cabinet checkingeach for packaging damage and expiration dates. When handling syringes,serological pipets, sterile forceps, scalpels, tissue plates, andneedles, make sure not to touch any surface that will come in contactwith the sterile product. Only the exterior of the syringe barrel,tubing, plunger tip and/or needle cap or sheath may be safely handled.Discard supply if the surface has been touched or has touched anon-sterile surface.e. Record lot numbers and expiration dates (if applicable) of allreagents and supplies to be used.f. Receive the thawed vial by cleaning the vial with lint-free wipemoistened with 70% alcohol before transferring into the biosafetycabinet.g. Using an aspirating needle with a syringe, withdraw as much liquidfrom the vial. Avoid suctioning the tissue.h. Using sterile forceps, transfer the tissue into a sterile 100 mmpetri dish.i. Add an aliquot of 5 ml rinse medium to the tissue fragments.j. Swirl the contents for 15-30 seconds, then remove the rinse mediumwith a pipette or syringe with aspirating needle. Repeat this rinseprocess twice.k. Add 2 mL of rinse medium to the tissue to avoid drying out thetissue.

2.4. Initiating MSC Outgrowth from Tissue:

a. Label the bottom of a 6-well plate “Outgrowth 1” with MSC lot numberor umbilical cord tissue ID and the date outgrowth is initiated. If 60mm tissue culture dish is used, divide the plate into 4 quadrants bydrawing a grid on the bottom of the dish.

b. Using sterile, disposable forceps, place one 3×3 mm to 5×5 mm tissueinto each well. If using a 60 mm tissue culture dish, place the tissueinto the middle of each quadrant to keep the tissues apart (more than 1cm from each other).

c. Fill each well with 3 ml of PTT6.

d. Using an aspirating needle coupled to 30 ml syringe, withdraw enoughmedia to barely cover the tissue. Do not tilt the plate. Do not touchthe bottom of the well with the aspirating needle.

e. Using an inverted light microscope, observe for cell outgrowth everyday (24±6 hrs). Real time cell culture imaging system may be used inplace of the light microscope.

f. Change media every day. Be sure to equilibrate the media to roomtemperature before use.

i. Aspirate off the medium.

ii. Add 3 ml of PTT6.

iii. Aspirate until tissue is barely submerged in the medium.

g. When cellular outgrowth is observed from the tissue, transplant thetissue to a new 6-well plate using the same procedure as 4.a to 4.eabove except label the plate “Outgrowth 2”. Maintain cell outgrowth in“Outgrowth 1” plate by adding 2 ml of PTT6 to each well. Observe forconfluency every day. Replace media every 2-3 days (be sure toequilibrate the media to room temperature before use).h. When cell outgrowth is observed in “Outgrowth 2” plate, repeat step4.a to 4.e except label the plate “Outgrowth 3.” Maintain cell outgrowthin “Outgrowth 2” plate by adding 2 ml of PTT6 to each well. Observe forconfluency every day. Replace media every 2-3 days (be sure toequilibrate the media to room temperature before use).i. When outgrowth is observed in “Outgrowth 3” plate, discard thetissue. If the tissues are very small and do not seem to interfere withcell growth, dispose of the tissue when subculturing.j. When cells reach 40-50% confluency, observe cells every days toprevent over-expansion.k. When cells reach 70-80% confluency, subculture the cells. Do notallow cells to expand beyond 80% confluence.

With the size of the tissue explants being about 1-3 mm, and the tissueexplant/cell culture is performed in 175 mm squared culture dishes, theaverage number of mesenchymal stem cells harvested from an explant istypically about 4,000-6,000 cells/explant. Accordingly, when themesenchymal stem cells are simultaneously grown out of 48 explants about300,000 cells can be obtained at harvest. These 300,000 mesenchymal stemcells collected from explants can then be used for subculturing byseeding a 175 cm² cell culture flask with such 300,000 cells asdescribed in the following Example 2.5 (this can be referred to asPassage 1). The mesenchymal stem cells obtained from this passage 1 canthen be used to seed again 175 cm² flasks (Passage 2) and expand thecells as described in the following Example 2.5. The cells obtained fromboth Passage 1 and Passage 2 can be “banked” by cryo-preservation, withthe mesenchymal stem cells obtained after Passage 2 being considered torepresent the Master Cell Bank which will be for further expansion ofthe mesenchymal stem cells, for example, in a bioreactor as explainedbelow in Example 2.7.

2.5. Subculturing MSC in Cell Culture Dishes

a. Perform viable particle while working in the biosafety cabinet.Equilibrate all media to room temperature before use.

b. When cell outgrowth reaches about 70-80% confluency, subculturecells.

i. Remove PTT6 from the petri dish.

ii. Rinse with HBSS without Calcium or Magnesium.

iii. Add 0.2 ml 1× TrypLE-EDTA and swirl for 1-2 minutes.

iv. Tilt the dish 30-45° to allow cells to shift down by gravitationalflow. Gentle tapping on the side of the plate expedites detachment.

v. Add 1 ml of PTT6. Pipette up and down gently then transfer cells to a15 ml centrifuge tube. Use clean pipette tip with each well. Cells fromall 6 wells can be pooled into a single 15 ml tube.

vi. Centrifuge for 10 minutes at 1200 rpm.

vii. Remove supernatant and resuspend cells with 5 ml PTT6.

c. Subculturing MSC

i. Aliquot 50 μl of the cell suspension and assay for TNC and viabilityby Trypan Blue Exclusion Assay.

ii. Count cells using a hemocytometer. Expect to count 20-100cells/square. If the count higher than 100, dilute the original sample1:5 and repeat Trypan Blue method using a hemocytometer.

iii. Calculate viable cells/ml and total viable cells:Viable cells/ml=viable cell count×dilution factor×10⁴  1.Total viable cells=viable cell count×dilution factor×totalvolume×10⁴  2.

iv. Calculate % viability:% viability=viable cell count×100/(viable cell count+dead cellcount)  1.

v. Dilute the cell suspension to 1.0×10⁶ cells/ml:“X” volume=Total viable cells/10⁶ cells/ml  1.For example, if total viable cell number is 1.0×10⁷;  2.“X”=10⁷/10⁶ cells/ml or 10 ml, therefore, you would bring your totalcell volume  3.up to 10 ml by adding 5 ml to your cell suspension (that is at 5 ml).

vi. If the cell suspension is less than 106/ml, determine the volumerequired to seed 2×10⁶ cells for each 150 mm petri dish or 175 cm2flask.Volume for 2×10⁶ cells=2×10⁶ cells÷viable cells/ml  1.For example, if viable cells/ml is 8×10⁵ cells/ml, 2×10⁶ cells÷8×10⁵cells/ml or 2.5 ml are needed.  2.

vii. Set aside 0.5 ml for MSC marker analysis.

viii. Seed 2×10⁶ cells to each 150 mm petri dish or 175 cm² flask with30 ml PTT6.

ix. Observe cells for attachment, colony formation, and confluence everythree days. When cells reach 40-50% confluence, observe cells everyone-two days to prevent over-expansion. DO NOT allow cells to expandbeyond 80% confluence. A real time cell culturing monitoring system canbe used in place of the light microscope.

x. Replace media every 2-3 days.

2.6 Cryopreserving MSC Cells

a. Perform viable particle while working in the biosafety cabinet.

b. When cells reach 70-80% confluence, detach cells using 2 ml 1×TrypLE-EDTA for each 150 mm petri dish or 175 cm2 flask.

i. Remove PTT6 from the petri dish.

ii. Wash with 5 ml HBSS or PBS without calcium or magnesium.

iii. Add 2 ml 1× TrypLE-EDTA and swirl for 1-2 minutes.

iv. Tilt the dish 30-45° to allow cells to shift down by gravitationalflow. Gentle tapping on the side of the petri dish helps to expeditedetachment.

v. Add 10 ml PTT6 to inactivate TrypLE. Mix well to dissociate cellclumps.

vi. Transfer cells to 15 ml centrifuge tube using a Pasteur pipette.

vii. Centrifuge for 10 minutes at 1200 rpm.

viii. Aspirate medium and resuspend with 10 ml PTT6.

ix. Aliquot 50 μl and determine total viable cell number and % viabilityas above. Cell count will need to be performed within 15 minutes as thecells may start clumping.

c. Preparing Cells for Cryopreservation

i. Prepare Cell Suspension Media and Cryopreservation Media and freezethe cells

2.7. Subculturing (Expansion) of MSC in a Quantum Bioreactor (TerumoBTC, Inc.)

It is also possible to use a Quantum Bioreactor can used to expand theMSC. The starting cell number for the expansion in the QuantumBioreactor should range between 20 to 30 million cells per run. Thetypical yield per run is 300 to 700 million MSC at harvest. TheBioreactor is operated following the protocol of the manufacturer. Theso obtained mesenchymal stem cells are typically cryo-preserved (seebelow) and serve as Working Cell Bank.

MATERIALS/REAGENTS:

1. Quantum Expansion Set

2. Quantum Waste Bag

3. Quantum Media Bag

4. Quantum Inlet Bag

5. PTT6

6. PBS

7. Fibronectin

8. TrypLE

9. 3 ml syringe

10. Glucose test strips

11. Lactate test strips

12. 60 ml Cell Culture Plate or equivalent

13. Medical Grade 5% CO₂ Gas-mix

14. 50 ml Combi-tip

EQUIPMENT:

1. Biosafety Cabinet

2. Glucose Meter (Bayer Healthcare/Ascensia Contour Blood Glucose Meter)

3. Lactate Plus (Nova Biomedical)

4. Peristaltic pump with head

5. Centrifuge, Eppendorf 5810

6. Sterile Tube Connector

7. M4 Repeat Pipettor

8. RF Sealer

PROCEDURE:

1. Preparing the Quantum Bioreactor

a) Priming the Quantum Bioreactor

b) Coating the bioreactor:

-   -   1) Prepare the fibronectin solution in the biosafety cabinet.        -   1) Allow lyophilized fibronectin to acclimate to room            temperature (≥15 min at room temperature)        -   2) Add 5 ml of sterile distilled water; do not swirl or            agitate        -   3) Allow fibronectin to go into solution for 30 min.        -   4) Using a 10 ml syringe attached with an 18g needle,            transfer the fibronectin solution to a Ccell inlet bag            containing 95 ml of PBS.    -   2) Attach the bag to the “reagent” line    -   3) Check for bubbles (bubbles may be removed by using “Remove IC        Air” or “Remove EC Air” and using “Wash” as the inlet source.    -   4) Open or set up program for coating the bioreactor (FIG. 1.        Steps 3-5).    -   5) Run the program    -   6) While the program is running to coat the bioreactor, prepare        a media bag with 4 L of PTT6 media.    -   7) Attach the media bag to the IC Media line using a sterile        tube connector.    -   8) When the bioreactor coating steps are completed, detach the        cell inlet bag used for fibronectin solution using a RF sealer.

c) Washing off excess fibronectin

d) Conditioning the bioreactor with media

2. Culturing the Cells in the Quantum Bioreactor

a) Loading and attaching the cells with Uniform Suspension:

b) Feeding and cultivation of the cells

-   -   1) Chose media flow rate to feed the cells.    -   2) Sample for lactate and glucose everyday.    -   3) Adjust the flow rate of the media as the lactate levels        increase. The actual maximal tolerable lactate concentration        will be defined by a flask culture from which the cells        originate. Determine if adequate PTT6 media is in the media bag.        If necessary, replace the PTT6 media bag with a fresh PTT6 media        bag.    -   4) When the flow rate has reached the desired value, measure        lactate level every 8-12 hours. If the lactate level does not        decrease or if the lactate level continues to increase, harvest        the cells.        3. Harvesting the Cells from the Quantum Bioreactor    -   a) When lactate concentration does not decrease, harvest the        cells after sampling for lactate and glucose for the last time.    -   b) Harvesting the cells:        -   1) Attach cell inlet bag filled with 100 ml TrypLE to the            “Reagent” line using a sterile tube connector.        -   2) Confirm sufficient PBS is in the PBS bag. If not, attach            a new bag with at least 1.7 liters of PBS to the “Wash” line            using a sterile tube connector.        -   3) Run the Harvest program            4. Cryopreserving the Cells    -   1) Once the cells have been harvested, transfer the cells to 50        ml centrifuge tube to pellet the cells.    -   2) Resuspend using 25 ml of cold cell suspension solution. Count        the cells using Sysmex or Biorad Cell counter. Attach the cell        count report to the respective Quantum Processing Batch Record.    -   3) Adjust cell concentration to 2×10⁷/ml    -   4) Add equal volume cryopreservation solution and mix well (do        not shake or vortex)    -   5) Using a repeat pipettor, add 1 ml of the cell suspension in        cryopreservative to each 1.8 ml vial. Cryopreserve using the CRF        program as described in the SOP D6.100 CB Cryopreservation Using        Controlled Rate Freezers    -   6) Store the vials in a designated liquid nitrogen storage        space.    -   7) Attach the CRF run report to the form respective MSC        P3-Quantum Processing Batch Record.

3. Analysis of Stem Cell Marker Expression in Mesenchymal Cord LiningStem Populations Isolated from Umbilical Cord Tissue, Using DifferentCulture Media

Flow cytometry experiments were carried out to to analyse mesenchymalstem cells isolated from the umbilical cord for the expression of themesenchymal stem cell markers CD73, CD90 and CD105.

For these experiments, mesenchymal stem cells were isolated fromumbilical cord tissue by cultivation of the umbilical cord tissue inthree different cultivation media, followed by subculturing of themesenchymal stem cells in the respective medium as set forth in Example2.

The three following culture media were used in these experiments: a) 90%(v/v/DMEM supplemented with 10% FBS (v/v), b) the culture medium PTT-4described in US patent application 2006/0078993 and the correspondingInternational patent application WO2006/019357 that consist of 90% (v/v)CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of WO2006/019357 andc) the culture medium of the present invention PPT-6 the composition ofwhich is described herein. In this flow cytometry analysis, twodifferent samples of the cord lining mesenchymal stem cell (CLMC)population were analysed for each of the three used culture media.

The following protocol was used for the flow cytometry analysis.

Materials and Methods

Instruments name Company Name Serial Name BD FACS CANDO BD V07300367Inverted Microscope, CKX41SF Olympus 4K40846 Centrifuge, Micro spinTabletop Biosan 010213-1201-0003

Reagent list Company Name CatLog Number 10 X Trypsin Biowest X0930-10010 X PBS Lonza 17-517Q DMEM Lonza 12-604F Fetal Bovine Serum GEhealthcare A11-151

Antibody list Company Name CatLog Number Human CD73 Purified AD2 0.1 mgBD 550256 Human CD90 Purified 5E10 1 mL BD 550402 Human CD105 Purified266 0.1 mg BD 555690 Alexa Fluor 647 goat anti-mouse IgG BD A21235 (H +L) *2 mg/mL*

Reagents name Composition 1 X PBS (1L) 100 ml of 10 X PBS + 900 ml ofsterile distilled H20 1x PBA (50 ml) 49.5 ml of 1XPBS + 0.5 ml of FBS

Procedure

a) Cell Isolation and Cultivation from the Umbilical Cord LiningMembrane

-   -   1. Explant tissue samples were incubated in a cell culture plate        and submerged in the respective medium, then keep it in CO₂        incubator at 37° C. as explained in Example 2.    -   2. The medium was changed every 3 days.    -   3. Cell outgrowth from tissue culture explants was monitored        under light microscopy.    -   4. At a confluence of about 70%, cells were separated from dish        by trypsinization (0.0125% trypsin/0.05% EDTA) and used for flow        cytometry experiments.

b) Trypsinization of Cells for Experiments

-   -   1. Remove medium from cell culture plate    -   2. Gently rinse with sterile 1×PBS to remove traces of FBS as        FBS will interfere with the enzymatic action of trypsin.    -   3. Add 1× trypsin to cell culture plate and incubate for 3-5 min        in 37° C.    -   4. Observe cells under microscope to ensure that they are        dislodged. Neutralize trypsin by adding complete media        containing FBS (DMEM with 10% FBS).    -   5. Use a pipette to break up cell clumps by pipetting cells in        media against a wall of the plate. Collect and transfer cell        suspension into 50 ml centrifuge tubes    -   6. Add sterile 1×PBS to plate and rinse it, Collect cell        suspension into the same centrifuge tube.    -   7. Centrifuge it at 1800 rpm for 10 mins.    -   8. Discard supernatant and re-suspend cell pellet with PBA        medium.

c) Counting Cells

-   -   1. Ensure that the haemocytometer and its cover slip are clean        and dry, preferably by washing them with 70% ethanol and letting        them dry before wiping them with Kim wipes (lint-free paper).    -   2. Aliquot a small amount of cells in suspension into a micro        centrifuge tube and remove from the BSC hood.    -   3. Stain cells in suspension with an equal volume of Trypan        Blue, e.g. to 500 μl of suspension add 500 μl of Trypan Blue        (dilution factor=2×, resulting in 0.2% Trypan Blue solution).    -   4. Avoid exposure of cells to Trypan Blue for longer than 30        mins as Trypan Blue is toxic and will lead to an increase in        non-viable cells, giving a false cell count.    -   5. Add 20 μl of the cell suspension mixture to each chamber of a        haemocytometer and view under a light microscope.        -   a. Count the number of viable cells (bright cells;            non-viable cells take up Trypan Blue readily and thus are            dark) in each quadrant of the haemocytometer for a total of            8 quadrants in the upper and lower chamber.            -   Total cell count is given as (Average number of                cells/quadrant)×10⁴ cells/ml.

d) Staining Cells

i. Preparation Before Staining Cells

-   -   Cell suspension are aliquot into 3 tubes (CD73, CD90, CD105) in        duplicates and 2 tubes (negative control), each containing        50,000 cells.

ii. Staining with Primary Antibody (Ab)

-   -   Add 1 μl [0.5 mg/ml Ab] of primary antibody to 100 ul cell        suspension. Incubate at 4° C. for 45 min.    -   Make up to 1 ml with PBA.    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Add 1 ml of PBA and re-suspend pellet    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Re-suspend in 100 ul PBA.

iii. Staining with secondary Ab—in the dark

-   -   Add 1 ul [0.5 mg/ml ab] of secondary antibody to 100 ul cell        suspension. Incubate at 4° C. for 30 min.    -   Make up to 1 ml with PBA.    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Add 1 ml of PBA and re-suspend pellet    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant    -   Re-suspend in 200-300 ul PBA for flow cytometry analysis    -   Transfer cells to FACS tube for reading in BD FACS CANDO flow        cytometry.

The results of the flow cytometry analysis are shown in FIG. 6a to FIG.6c . FIG. 6a shows the percentage of isolated mesenchymal cord liningstem cells expressing stem cell markers CD73, CD90 and CD105 afterisolation from umbilical cord tissue and cultivation in DMEM/10% FBS,FIG. 6b shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-4 and FIG. 6c showsthe percentage of isolated mesenchymal cord lining stem cells expressingstem cell markers CD73, CD90 and CD105 after isolation from umbilicalcord tissue and cultivation in PTT-6. As can be seen from FIG. 6a , thepopulation isolated using DMEM/10% FBS as culture medium cultivation hasabout 75% CD73+ cells, 78% 90+ cells and 80% CD105+ cells (average oftwo experiments), while after isolation/cultivation of umbilical cordtissue using PPT-4 culture medium (see FIG. 6b ) the number ofmesenchymal stem cells that are CD73-positive, CD90-positive andCD105-positive are about 87% (CD73+ cells), 93%/CD90+ cells) and 86%(CD105+ cells) average of two experiments. The purity of the mesenchymalstem cell population that was obtained by means of cultivation in thePTT-6 medium of the present invention is at least 99.0% with respect toall three markers (CD73, CD90, CD105), meaning the purity of this cellpopulation is significant higher than for cultivation using PPT-4 mediumor DMEM/10% FBS. In addition and even more importantly, the mesenchymalstem cell population obtained by means of cultivation in PTT-6 isessentially a 100% pure and defined stem cell population. This makes thestem cell population of the present invention the ideal candidate forstem cell based therapies. Thus, this population of mesenchymal cordlining stem cells may become the gold standard for such stem cell basedtherapeutic approaches.

The findings shown in FIG. 6 are further corroborated by the results ofthe flow cytometry analysis that are shown in FIG. 7a and FIG. 7b . FIG.7a shows the percentage of isolated mesenchymal cord lining stem cells(mesenchymal stem cells of the amniotic membrane of umbilical cord) thatexpress the stem cell markers CD73, CD90 and CD105 and lack expressionof CD34, CD45 and HLA-DR after isolation from umbilical cord tissue andcultivation in PTT-6 medium. As shown in FIG. 7a , the mesenchymal stemcell population contained 97.5% viable cells of which 100% expressedeach of CD73, CD90 and CD105 (see the rows “CD73+CD90+” and“CD73+CD105+”) while 99.2% of the stem cell population did not expressCD45 and 100% of the stem cell population did not express CD34 andHLA-DR (see the rows “CD34−CD45− and “CD34−HLA-DR−). Thus, themesenchymal stem cells population obtained by cultivation in PTT-6medium is essentially a 100% pure and defined stem cell population thatmeets the criteria that mesenchymal stem cells are to fulfill to be usedfor cell therapy (95% or more of the stem cell population express CD73,CD90 and CD105, while 98% or more of the stem cell population lackexpression of CD34, CD45 and HLA-DR, see Sensebe et al. “Production ofmesenchymal stromal/stem cells according to good manufacturingpractices: a review”, supra). It is noted here that the presentmesenchymal stem cells of the amniotic membrane are adhere to plastic instandard culture conditions and differentiate in vitro into osteoblasts,adipocytes and chondroblasts, see U.S. Pat. Nos. 9,085,755, 8,287,854 orWO2007/046775 and thus meet the criteria generally accepted for use ofmesenchymal stem cells in cellular therapy.

FIG. 7b shows the percentage of isolated bone marrow mesenchymal stemcells that express CD73, CD90 and CD105 and lack expression of CD34,CD45 and HLA-DR. As shown in FIG. 7b , the bone marrow mesenchymal stemcell population contained 94.3% viable cells of which 100% expressedeach of CD73, CD90 and CD105 (see the rows “CD73+CD90+” and“CD73+CD105+”) while only 62.8% of the bone marrow stem cell populationlacked expression of CD45 and 99.9% of the stem cell population lackedexpression CD34 and HLA-DR (see the rows “CD34−CD45− and “CD34-HLA-DR−).Thus, the bone marrow mesenchymal stem cells that are considered to begold standard of mesenchymal stem cells are by far less homogenous/purein terms of stem cell marker than the mesenchymal stem cells population(of the amniotic membrane of the umbilical cord) of the presentapplication. This finding also shows that the stem cell population ofthe present invention may be the ideal candidate for stem cell basedtherapies and may become the gold standard for stem cell basedtherapeutic approaches.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention. Theinvention has been described broadly and generically herein. Each of thenarrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Further embodiments of the invention willbecome apparent from the following claims.

The invention is further characterized by the following items:

1. A method of isolating a mesenchymal stem cell population from theamniotic membrane of the umbilical cord, the method comprisingcultivating umbilical cord tissue in a culture medium comprising DMEM(Dulbecco's modified eagle medium), F12 (Ham's F12 Medium), M171 (Medium171) and FBS (Fetal Bovine Serum).2. The method of item 1, wherein the culture medium comprises DMEM in afinal concentration of about 55 to 65% (v/v), F12 in a finalconcentration of about 5 to 15% (v/v), M171 in a final concentration ofabout 15 to 30% (v/v) and FBS in a final concentration of about 1 to 8%(v/v).3. The method of item 2, wherein the culture medium comprises DMEM in afinal concentration of about 57.5 to 62.5% (v/v), F12 in a finalconcentration of about 7.5 to 12.5% (v/v), M171 in a final concentrationof about 17.5 to 25.0% (v/v) and FBS in a final concentration of about1.75 to 3.5% (v/v).4. The method of item 3, wherein the culture medium comprises DMEM in afinal concentration of about 61.8% (v/v), F12 in a final concentrationof about 11.8% (v/v), M171 in a final concentration of about 23.6% (v/v)and FBS in a final concentration of about 2.5% (v/v).5. The method of any of items 1 to 4, wherein the culture medium furthercomprises Epidermal Growth Factor (EGF) in a final concentration ofabout 1 ng/ml to about 20 ng/ml.6. The method of any of items 1 to 5, wherein the culture mediumcomprises EGF in a final concentration of about 10 ng/ml.7. The method of any of items 1 to 6, wherein the culture mediumcomprises Insulin in a final concentration of about 1 μg/ml to 10 μg/ml.8. The method of any of items 1 to 7, wherein the culture mediumcomprises Insulin in a final concentration of about 5 μg/ml.9. The method of any of the foregoing items, wherein the culture mediumfurther comprises at least one of the following supplements: adenine,hydrocortisone, and 3,3′,5-Triiodo-L-thyronine sodium salt (T3).10. The method of any of the foregoing items, wherein the culture mediumcomprises all three of adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3).11. The method of item 11 or 12, wherein the culture medium comprisesadenine in a final concentration of about 0.01 to about 0.1 μg/mladenine, hydrocortisone in a final concentration of about 0.1 to about10 μg/ml hydrocortisone and/or 3,3′,5-Triiodo-L-thyronine sodium salt(T3) in a final concentration of about 0.5 to about 5 ng/ml.12. The method of any of the foregoing items, comprising culturing theumbilical cord tissue till the cell outgrowth of the mesenchymal stemcells of the amniotic membrane reaches about 70-80% confluency.13. The method of item 12, comprising removing the mesenchymal stemcells from the cultivation container used for the cultivation.14. The method of item 13, wherein removing the mesenchymal stem cellsfrom the cultivation container is carried out by enzymatic treatment.15. The method of item 14, wherein the enzymatic treatment comprisestrypsination.16. The method of any of items 13 to 15, wherein the mesenchymal stemcells are transferred for subculturing to a cultivation container forsubculturing.17. The method of item 16, wherein the mesenchymal cells are suspendedfor subculturing at a concentration 1.0×10⁶ cells/ml.18. The method of item 17, wherein the mesenchymal stem cells aresubcultured in a culture medium as defined in any of the items 1 to 10.19. The method of item 18, wherein the mesenchymal stem cells aresubcultured till the mesenchymal stem cells reach about 70-80%confluency.20. The method of any of items 16 to 19, wherein the subculturing iscarried out in a self-contained bioreactor.21. The method of item 20, wherein the bioreactor is selected from thegroup consisting of a parallel-plate bioreactor, a hollow-fiberbioreactor and and a micro-fluidic bioreactor.22. The method of any of the foregoing items, wherein the umbilical cordtissue is a piece of the entire umbilical cord or the amniotic membraneof the umbilical cord.23. The method of any of the foregoing items wherein cultivation iscarried out in a CO₂ cell culture incubator at a temperature 37° C.24. The method of item 23, comprising removing the mesenchymal stemcells from the cultivation container used for the subcultivation.25. The method of item 24, wherein removing the mesenchymal stem cellsfrom the cultivation container is carried out by enzymatic treatment.26. The method of item 25, wherein the enzymatic treatment comprisestrypsination.27. The method of item 26, further comprising collecting the isolatedmesenchymal stem cells.28. The method of any of the foregoing items, wherein at least about 90%or more, about 91% or more, about 92% or more, about 92% or more, about93% or more, about 94% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more about 99% or more of the isolatedmesenchymal stem cells express the following markers: CD73, CD90 andCD105.29. The method of any of the foregoing items, wherein at least about 90%or more, about 91% or more, about 92% or more, about 92% or more, about93% or more, about 94% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more about 99% or more of the isolatedmesenchymal stem cells lack expression of the following markers: CD34,CD45 and HLA-DR (Human Leukocyte Antigen—antigen D Related).30. The method of any of items 28 or 29, wherein about 97% or more,about 98% or more about 99% or more of the isolated mesenchymal stemcells express CD73, CD90 and CD105 and lack expression of CD34, CD45 andHLA-DR.31. The method of any of the foregoing items, further comprisingpreserving the isolated stem/progenitor cells for further use.32. The method of item 31, wherein preserving is carried out bycryo-preservation.33. An isolated mesenchymal stem population of the amniotic membrane ofthe umbilical cord, wherein at least about 90% or more cells of the stemcell population express each of the following markers: CD73, CD90 andCD105.34. The mesenchymal stem cell population of item 33, wherein least about90% or more cells of the stem cell population lack expression of thefollowing markers: CD34, CD45 and HLA-DR.35. The mesenchymal stem cell population of item 34, wherein at leastabout 91% or more, about 92% or more, about 92% or more, about 93% ormore, about 94% or more, about 95% or more, about 96% or more, about 97%or more, about 98% or more about 99% or more cells of the isolatedmesenchymal stem cell population express each of CD73, CD90 and CD105and lack expression of each of CD34, CD45 and HLA-DR.36. The mesenchymal stem cell population of any of items 33 to 35,wherein the population is obtainable by the method as defined in any ofitems 1 to 30.37. The mesenchymal stem cell population of any of items 33 to 35,wherein the population is obtained by the method as defined in any ofitems 1 to 30.38. A pharmaceutical composition comprising an isolated mesenchymal stempopulation of the amniotic membrane of the umbilical cord, wherein atleast about 90% or more cells of the stem cell population express eachof the following markers: CD73, CD90 and CD105 and lack expression ofeach of the following markers: CD34, CD45 and HLA-DR.39. The pharmaceutical composition of item 38, wherein thepharmaceutical composition is adapted for systemic or topicalapplication.40. The pharmaceutical composition of item 38 or 39, further comprisinga pharmaceutically acceptable excipient.41. A method of making a culture medium suitable for isolating amesenchymal stem cell population from the amniotic membrane of theumbilical cord, the method comprising, mixing to obtain a final volumeof 500 ml culture medium:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) (final concentration of 2.5%)

42. The method of item 41, further comprising adding

v. 1 ml EGF stock solution (5 μg/ml) to achieve a final concentration of10 ng/ml)

vi. Insulin 0.175 ml stock solution (14.28 mg/ml) to achieve a finalconcentration of 5 μg/ml.

43. The method of item 41 or 42, further comprising adding to DMEM oneor more of the following supplements: adenine, hydrocortisone,3,3′,5-Triiodo-L-thyronine sodium salt (T3), thereby reaching a totalvolume of 500 ml culture medium.

44. The method of item 43, wherein the final concentration of thesupplements in DMEM are as follows.

about 0.05 to 0.1 μg/ml adenine, for example about 0.025 μg/ml adenine,

about 1 to 10 μg/ml hydrocortisone,

about 0.5 to 5 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3), forexample 1.36 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3).

45. A cell culture medium obtainable by the method of any of items 41 to44.

46. A method of isolating mesenchymal stem cells from the amnioticmembrane of the umbilical cord, comprising cultivating amniotic membranetissue in the culture medium prepared by the method as defined in any ofitems 41 to 44.

47. A cell culture medium comprising:

DMEM in the final concentration of about 55 to 65% (v/v),

F12 in a final concentration of about 5 to 15% (v/v),

M171 in a final concentration of about 15 to 30% (v/v) and

FBS in a final concentration of about 1 to 8% (v/v).

48. The cell culture medium of item 47, wherein the culture mediumcomprises DMEM in the final concentration of about 57.5 to 62.5% (v/v),F12 in a final concentration of about 7.5 to 12.5% (v/v), M171 in afinal concentration of about 17.5 to 25.0% (v/v) and FBS in a finalconcentration of about 1.75 to 3.5% (v/v).49. The cell culture medium of item 48, wherein the culture mediumcomprises DMEM in a final concentration of about 61.8% (v/v), F12 in afinal concentration of about 11.8% (v/v), M171 in a final concentrationof about 23.6% (v/v) and FBS in a final concentration of about 2.5%(v/v).50. The cell culture medium of any of items 47 to 49, wherein theculture medium further comprises Epidermal Growth Factor (EGF) in afinal concentration of about 1 ng/ml to about 20 ng/ml.51. The cell culture medium of any of items 7 to 50, wherein the culturemedium comprise EGF in a final concentration of about 10 ng/ml.52. The cell culture medium of any of items 47 to 51, wherein theculture medium comprises Insulin in a final concentration of about 1μg/ml to 10 mg/ml.53. The cell culture medium of item 52, wherein the culture mediumcomprises Insulin in a final concentration of about 5 μg/ml.54. The cell culture medium of any of items 47 to 53, wherein theculture medium further comprises at least one of the followingsupplements: adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyroninesodium salt (T3).55. The cell culture medium of item 54, wherein the culture mediumcomprises all three of adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3).56. The cell culture medium of item 54 or 55, wherein the culture mediumcomprises adenine in a final concentration of about 0.05 to about 0.1mg/ml adenine, hydrocortisone in a final concentration of about 1 toabout 10 μg/ml hydrocortisone and/or 3,3′,5-Triiodo-L-thyronine sodiumsalt (T3) in a final concentration of about 0.5 to about 5 ng/ml.57. The cell culture medium of any of items 47 to 56, wherein 500 ml ofthe cell culture medium comprise:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) (final concentration of 2.5%)

58. The cell culture medium of item 57, further comprising

v. EGF in a final concentration of 10 ng/ml

vi. Insulin in a final concentration of 5 μg/ml.

vi. Insulin 0.175 ml (final concentration of 5 μg/ml)

59. The cell culture medium of item 57 or 58, further comprising adeninein a final concentration of about 0.05 to about 0.1 μg/ml adenine,hydrocortisone in a final concentration of about 1 to about 10 mg/mlhydrocortisone and/or 3,3′,5-Triiodo-L-thyronine sodium salt (T3) in afinal concentration of about 0.5 to about 5 ng/ml.60. The use of a cell culture medium as defined in any of items 47 to 59for isolation of mesenchymal stem cells from the amniotic membrane ofumbilical cord.61. The use of a cell culture medium as defined in any of items 47 to 59for cultivation of mesenchymal stem cells from the amniotic membrane ofumbilical cord.

What is claimed is:
 1. An isolated mesenchymal stem population of theamniotic membrane of the umbilical cord, wherein cells of the stem cellpopulation express the POU5f1 gene, and wherein at least about 97% ormore cells of the stem cell population express each of the followingmarkers: CD73, CD90 and CD105 and lack expression of the followingmarkers: CD34, CD45 and HLA-DR.
 2. A pharmaceutical compositioncomprising an isolated mesenchymal stem population of the amnioticmembrane of the umbilical cord, wherein cells of the stem cellpopulation express the POU5f1 gene, and wherein at least about 97% ormore cells of the stem cell population express each of the followingmarkers: CD73, CD90 and CD105 and lack expression of each of thefollowing markers: CD34, CD45 and HLA-DR.
 3. The mesenchymal stem cellpopulation of claim 1, wherein least about 98% cells of the stem cellpopulation express each of CD73, CD90 and CD105 and lack expression ofthe following markers: CD34, CD45 and HLA-DR.
 4. The mesenchymal stemcell population of claim 1, wherein least about 99% cells of the stemcell population express each of CD73, CD90 and CD105 and lack expressionof the following markers: CD34, CD45 and HLA-DR.
 5. The pharmaceuticalcomposition of claim 2, wherein at least about 98% cells of the stemcell population express each of the following markers: CD73, CD90 andCD105 and lack expression of each of the following markers: CD34, CD45and HLA-DR.
 6. The pharmaceutical composition of claim 2, wherein atleast about 99% cells of the stem cell population express each of thefollowing markers: CD73, CD90 and CD105 and lack expression of each ofthe following markers: CD34, CD45 and HLA-DR.